Academic literature on the topic 'SR Protein Specific Kinase 2'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the lists of relevant articles, books, theses, conference reports, and other scholarly sources on the topic 'SR Protein Specific Kinase 2.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Journal articles on the topic "SR Protein Specific Kinase 2"
1
Wang, Huan-You, Wen Lin, Jacqueline A. Dyck, Joanne M. Yeakley, Zhou Songyang, Lewis C. Cantley, and Xiang-Dong Fu. "SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells." Journal of Cell Biology 140, no. 4 (February 23, 1998): 737–50. http://dx.doi.org/10.1083/jcb.140.4.737.
Full textAbstract:
Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.
2
McKee, Charia, Dan J. Bare, and Xun Ai. "Stress-driven cardiac calcium mishandling via a kinase-to-kinase crosstalk." Pflügers Archiv - European Journal of Physiology 473, no. 3 (February 15, 2021): 363–75. http://dx.doi.org/10.1007/s00424-021-02533-2.
Full textAbstract:
AbstractCalcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca2+-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.
3
Karakama, Yuko, Naoya Sakamoto, Yasuhiro Itsui, Mina Nakagawa, Megumi Tasaka-Fujita, Yuki Nishimura-Sakurai, Sei Kakinuma, et al. "Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase." Antimicrobial Agents and Chemotherapy 54, no. 8 (May 24, 2010): 3179–86. http://dx.doi.org/10.1128/aac.00113-10.
Full textAbstract:
ABSTRACT Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.
4
Meldrum, D. R., J. C. Cleveland, M. B. Mitchell, B. C. Sheridan, F. Gamboni-Robertson, A. H. Harken, and A. Banerjee. "Protein kinase C mediates Ca2(+)-induced cardioadaptation to ischemia-reperfusion injury." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 271, no. 3 (September 1, 1996): R718—R726. http://dx.doi.org/10.1152/ajpregu.1996.271.3.r718.
Full textAbstract:
Although protein kinase C (PKC)-mediated cardioadaptation to ischemia-reperfusion (IR) is accompanied by increased intracellular Ca2+ concentration, it is unknown whether a preischemia sarcoplasmic reticulum (SR) Ca2+ release affects PKC-mediated post-IR functional protection. To study this, crystalloid-perfused (Langendorff) Sprague-Dawley rat hearts were used to assess the effects of a ryanodine (Ry)-induced preischemia Ca2+ load (Ry, 5 nM/2 min, retrograde coronary) 10 min before global IR (20 min). Ry was administered with and without each of two different PKC inhibitors (20 microM chelerythrine and 150 nM bisindolylmaleimide I-HCl). Ry improved myocardial functional recovery (developed pressure, end-diastolic pressure, coronary flow, and creatine kinase activity), which was eliminated after PKC inhibition. Immunohistochemical staining for PKC isoforms demonstrated that Ry induces specific PKC translocation of alpha-, delta-, and zeta-isoforms. We conclude that 1) a preischemia Ca2+ load from the SR results in post-IR myocardial functional protection 2) Ca(2+)-induced functional protection is PKC regulated via the translocation of specific isoforms, and 3) Ca(2+)-induced cardioadaptation to IR injury may have important therapeutic implications prior to planned ischemic events such as cardiac allograft preservation and cardiac bypass surgery.
5
Manna, Pulak R., Youngah Jo, and Douglas M. Stocco. "Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling." Journal of Endocrinology 193, no. 1 (April 2007): 53–63. http://dx.doi.org/10.1677/joe-06-0201.
Full textAbstract:
The steroidogenic acute regulatory (StAR) protein plays a central role in the regulation of steroid biosynthesis. While steroidogenesis is influenced by many processes, their modes of actions, in a few cases, remain obscure. In this study, we explored the mechanism of action of one such signaling pathway, the extracellular signal-regulated kinase 1/2 (ERK1/2), in regulating StAR expression and steroidogenesis in conjunction with the protein kinase A (PKA) and protein kinase C (PKC) pathways. Using MA-10 mouse Leydig tumor cells, we demonstrate that the activation of PKC and PKA signaling, by phorbol-12-myristate-13-acetate (PMA) and dibutyryl cAMP (dbcAMP)/human chorionic gonadotropin (hCG) respectively, was able to phosphorylate ERK1/2, an event markedly decreased by an upstream kinase inhibitor, U0126. Treatment with PMA enhanced StAR protein expression (associated with a slight increase in progesterone synthesis) but not its phosphorylation (P-StAR), which, in contrast, coordinately increased in response to dbcAMP/hCG. Inhibition of ERK1/2 activity by U0126 decreased PMA-treated StAR expression but increased dbcAMP/hCG-mediated StAR and P-StAR; however, progesterone levels were attenuated. U0126 was found to affect StAR expression and steroidogenesis both at the transcriptional and translational levels. Further studies demonstrated that the effect of U0126 on PMA- and dbcAMP/hCG-mediated StAR expression and steroid synthesis was tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). In fact, both DAX-1 and SR-B1 appear to play important roles in hormone-regulated steroidogenesis. These findings clearly demonstrate that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways and is stimulus specific in mouse Leydig cells.
6
Vasanji, Zainisha, Elliott J. F. Cantor, Danijel Juric, Mellissa Moyen, and Thomas Netticadan. "Alterations in cardiac contractile performance and sarcoplasmic reticulum function in sucrose-fed rats is associated with insulin resistance." American Journal of Physiology-Cell Physiology 291, no. 4 (October 2006): C772—C780. http://dx.doi.org/10.1152/ajpcell.00086.2005.
Full textAbstract:
Diabetes mellitus (DM) causes the development of a specific cardiomyopathy that results from the metabolic derangements present in DM and manifests as cardiac contractile dysfunction. Although myocardial dysfunction in Type 1 DM has been associated with defects in the function and regulation of the sarcoplasmic reticulum (SR), very little is known about SR function in Type 2 DM. Accordingly, this study examined whether abnormalities in cardiac contractile performance and SR function occur in the prestage of Type 2 DM (i.e., during insulin resistance). Sucrose feeding was used to induce whole body insulin resistance, whereas cardiac contractile performance was assessed by echocardiography and SR function was measured by SR calcium (Ca2+) uptake. Sucrose-fed rats exhibited hyperinsulinemia, hyperglycemia, and hyperlipidemia relative to control rats. Serial echocardiographic assessments in the sucrose-fed rats revealed early abnormalities in diastolic function followed by late systolic dysfunction and concurrent alterations in myocardial structure. The hearts of the 10-wk sucrose-fed rats showed depressed SR function demonstrated by a significant reduction in SR Ca2+ uptake. The decline in SR Ca2+ uptake was associated with a significant decrease in the cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of phospholamban. The results show that abnormalities in cardiac contractile performance and SR function occur at an insulin-resistant stage before the manifestation of overt Type 2 DM.
7
Allemand, Eric, Renata Gattoni, Henri-Marc Bourbon, James Stevenin, Javier F. Cáceres, Johann Soret, and Jamal Tazi. "Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation." Molecular and Cellular Biology 21, no. 4 (February 15, 2001): 1345–59. http://dx.doi.org/10.1128/mcb.21.4.1345-1359.2001.
Full textAbstract:
ABSTRACT The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5′ alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.
8
Damiani, E., and A. Margreth. "Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca2+- and low-density-lipoprotein-binding protein in rabbit skeletal muscle." Biochemical Journal 277, no. 3 (August 1, 1991): 825–32. http://dx.doi.org/10.1042/bj2770825.
Full textAbstract:
Skeletal-muscle sarcoplasmic reticulum (SR) comprises two distinct domains, corresponding to the free membrane of longitudinal SR (LSR) and the junctional membrane region of the terminal cisternae (TC), respectively. The junctional membrane contains the ryanodine receptor (RyR)/Ca(2+)-release channel and additional minor protein components that still require biochemical investigation, in relation to excitation-contraction coupling. Recent findings suggested the involvement in this process of a 170 kDa protein [Kim, Caswell, Talvenheimo & Brandt (1990) Biochemistry 29, 9281-9289], also characterized as a phosphoprotein in junctional TC in independent studies [Chu, Submilla, Inesi, Jay & Campbell (1990) Biochemistry 29, 5899-5905]. We show that this protein is a specific substrate of exogenous cyclic AMP-dependent protein kinase, that it is exposed to the outer surface of intact TC vesicles, and that it co-localizes with the RyR to the junctional membrane. Comparative analysis of LSR and TC subfractions for the 160 kDa glycoprotein sarcalumenin, using Western-blot techniques and specific monoclonal antibodies or concanavalin A as a ligand, revealed that the distribution of this protein within the SR corresponds inversely to both that of the RyR and of the 170 kDa protein. The 170 kDa protein, like sarcalumenin, stains blue with the cationic dye Stains-All and binds 45Ca2+ on blots, but it is uniquely distinguished by its ability to bind 125I-labelled low-density lipoprotein. The similarity of these properties, as well as the pI and solubility properties, to those described for the SR protein, recently purified and cloned and named histidine-rich Ca(2+)-binding protein [HCP; Hofmann, Brown, Lee, Pathak, Anderson & Goldstein (1989) J. Biol. Chem. 264, 8260-8270], makes it very likely that our protein and HCP may indeed be identical. The protein described in the present study differs from sarcalumenin because its migration in SDS/PAGE is accelerated in the presence of Ca2+, a previously reported property of other Ca(2+)-binding proteins [leMaire, Lund, Viel, Champeil & Moller (1989) J. Biol. Chem. 265, 1111-1123], arguing for Ca(2+)-induced protein-conformational changes. Kinase-dependent phosphorylation of our protein is another distinguishing feature, which, although not previously reported for HCP, is consistent with the presence of potential serine/threonine phosphorylation sites in the middle portion of the cloned HCP molecule. The finding that HCP, contrary to early views, selectively binds to the cytoplasmic side of the junctional membrane, together with its newly characterized properties, seem to provide new clues as to a possible role in electromechanical coupling and/or Ca2+ release.
9
Fujioka, Y., T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, M. Tsuda, T. Takada, and M. Kasuga. "A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion." Molecular and Cellular Biology 16, no. 12 (December 1996): 6887–99. http://dx.doi.org/10.1128/mcb.16.12.6887.
Full textAbstract:
Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of approximately 115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor- and cell adhesion-induced cell signaling.
10
Kuranaga, Yuki, Nobuhiko Sugito, Haruka Shinohara, Takuya Tsujino, Kohei Taniguchi, Kazumasa Komura, Yuko Ito, Tomoyoshi Soga, and Yukihiro Akao. "SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells." International Journal of Molecular Sciences 19, no. 10 (October 2, 2018): 3012. http://dx.doi.org/10.3390/ijms19103012.
Full textAbstract:
Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.
Dissertations / Theses on the topic "SR Protein Specific Kinase 2"
1
VIVARELLI, SILVIA. "New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2010. http://hdl.handle.net/10281/10747.
Full textAbstract:
The aim of the first work (presented in the Chapter 2) was to investigate the role performed by SRPK2 kinase in the regulation of alternative splicing in SH-SY5Y neuroblastoma cells after paraquat treatment (a complex I mitochondrial respiratory chain inhibitor). Alternative splicing is a versatile form of genetic control whereby a common precursor messenger RNA (pre-mRNA) is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. This process is particularly important in the nervous system and its essential nature is underscored by the finding that its misregulation is a common feature of human diseases, including neurodegenerative pathologies. Our approach to gain insight the regulation of neuron specific pre-mRNA splicing brought us to the characterization of SRPK2 kinase because this protein phosphorylates Serine/Arginine-rich domain (RS-domain)-containing proteins and it is expressed almost exclusively in the nervous system. In order to understand how SRPK2 intracellular localization and activity are regulated, in first instance we performed a mutational analysis. These mutants have been characterized by transient transfection in SH-SY5Y neuroblastoma cells. We analysed SRPK2 intracellular localization both under physiological condition and after generating stress through mitochondrial damage, since mitochondrial damage and oxidative stress are found in many neurodegenerative diseases. We also determined the effect of this stress treatment on SRPK2 phosphorylation and its nuclear translocation. We did this first by using the minigene E1A, an alternative splicing reporter system, and also by analysing both SR proteins intracellular localization and their phosphorylation status. This work showed that not only paraquat treatment increased the phosphorylated SRPK2 fraction, but also that a specific phosphorylation at its 581 residue could be connected with the nuclear translocation of SRPK2. Consequently, this nuclear translocation brought to a splicing change in the isoform ratio of the minigene reporter system. After the drug treatment we also observed a specific speckled enlarged pattern coupled with an increase in the phosphorylation level for the SR classical proteins, known targets of SR protein kinase 2. These findings supported a functional link between the nuclear translocation and the activity of this neuronal specific kinase.
In the second line of our research (presented in the Chapter 3) we performed experiments assessing the function of the mammalian 3’ end processing factor CFIm68 in the mRNA export, thus confirming its action as an adaptor for TAP/NXF1 mRNA export receptor. In particular I helped to demonstrate that the tethering of CFIm68 promoted mRNA export by designing and performing an RNA FISH assay. I used a RNA-biotinylated probe that detected the intracellular localisation of an mRNA reporter construct co-transfected with the CFIm68 protein or control proteins. Therefore we observed an increase of the probe fluorescent cytoplasmic signal only in the presence of the overexpressed CFIm68 but not with other control proteins, observation confirmed by further Real Time PCR data.
In the third line of our research (presented in the Chapter 4) we reported that CFIm68 was also involved in the 3’ end cleavage of mammalian histone transcripts (not polyadenylated) by interacting with the LSM11 U7 snRNP component both in vitro and in vivo thus increasing the efficiency of the 3’ end processing in vivo. In this context I performed the Bimolecular Fluorescence Complementation (BiFC) analysis. I co-transfected the CFIm68 and the LSM11 proteins (or its MPL loss-of-function mutant) fused respectively with the C-terminus and the N-terminus of a Venus-Yellow Fluorescent Protein. Thus I detected a nuclear fluorescent complementation in more than 90% of the cells (or not complementation with the MPL mutant counterpart). This data supported our whole characterized observation concerning the involvement of this mammalian cleavage factor in 3’ histone mRNAs processing.
2
Chatzis, George J. "Biochemical properties of the muscle-specific calcium(2+)/calmodulin-dependent protein kinase II beta isoform." Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9357.
Full textAbstract:
Cytosolic calcium (Ca2+) levels are critical for the control of muscle contraction and are tightly regulated by a variety of Ca 2+ transport systems localized in various membranes. Ca2+ binding proteins such as calmodulin (CaM) and Ca2+/CaM-dependent protein kinases (CaM Kinases) are believed to exert major regulatory control on Ca2+ activity. Previous studies in this lab led to the cloning of a cDNA encoding a CaM Kinase II beta isoform from skeletal muscle that differed from the classical beta isoform by the inclusion of three alternatively spliced exons in the variable domain which were enriched in proline residues. A CaM Kinase, assumed to be localized in the sarcoplasmic reticulum (SR), has been implicated in the regulation of excitation-contraction (E-C) coupling. We hypothesized that this novel CaM Kinase II beta isoform called SOCK (Son Of CaM Kinase) may be the CaM Kinase II isoform that regulates E-C coupling by being targeted to specific regions of the SR, whereby it phosphorylates critical Ca2+ transporting proteins such as the ryanodine (RyR) and dihydropyridine (DHPR) receptors in response to changes in Ca2+ levels. (Abstract shortened by UMI.)
3
Tangeman, Larissa J. "Targeted knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits." Wright State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=wright1323880762.
Full text
4
Törmänen, Persson Heidi. "The Adenovirus L4-33K Protein : A Key Regulator of Virus-specific Alternative Splicing." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-159632.
Full textAbstract:
Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection. The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation. We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing. In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.
5
Spain, V. "A direct interaction between the Parkinson's disease protein leucine-rich repeat kinase 2 and specific β-tubulin isoforms regulates tubulin acetylation." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1458026/.
Full textAbstract:
Mutations in LRRK2 are a common cause of Parkinson’s disease (PD). LRRK2 encodes leucine-rich repeat kinase 2 (LRRK2), a ROCO protein. It has an enzymatic core consisting of a Ras of complex proteins (Roc) GTPase domain and kinase domain, surrounded by protein-protein interaction regions. Pathogenic LRRK2 mutations modify activity in these enzymatic domains, but how this leads to neurodegeneration is still to be elucidated. One of the few confirmed LRRK2 interactors is tubulin, the main constituent of microtubules (MTs) and part of the cytoskeletal network. Disease-causing mutations in LRRK2 alter this network, reducing neurite outgrowth and leading to accumulation of hyperphosphorylated MT-associated protein (MAP) tau. Meanwhile changes in post-translational modifications of tubulin and MAPs alter the dynamic instability of MTs, leading to aberrant axonal transport, synaptic dysfunction and axonal degeneration. I investigated the LRRK2-tubulin interaction. Using yeast two-hybrid I demonstrated that the interaction is conferred by the LRRK2 Roc domain and the C-terminus of the β-tubulin isoforms TUBB, TUBB4 and TUBB6. The interaction requires Lys362 and Ala364 and is blocked in isoforms expressing a serine at these positions. This site is on the luminal face of MT protofibrils, close to the paclitaxel binding site and α-tubulin Lys40 acetylation site, both of which are involved in MT stability. This location is poorly accessible within mature, stabilised MTs but exposed in dynamic MT populations. Consistent with this finding, endogenous LRRK2 located to dynamic growth cone MTs in SH-SY5Y cells. Overexpression and knock-out studies in HEK cells and mouse embryonic fibroblasts showed that LRRK2 is associated with reduced α-tubulin acetylation. These results demonstrate the specificity of the LRRK2-tubulin interaction, suggesting LRRK2 distribution at the cytoskeleton is determined by the tubulin composition and may vary between cell types. Changes in MT acetylation in the presence of disease-causing LRRK2 mutations could contribute to pathogenic mechanisms, with altered MT stability implicated in PD neurodegeneration. As mutations affecting the β-tubulin C-terminal residues could disrupt the LRRK2 interaction without compromising MT integration, a cohort of late-onset familial PD cases was also screened for mutations within the cytoskeleton.
6
Cardin, Eric. "Function of Nck-1 adaptor protein as modulator of elF2alpha phosphorylation by specific elF2alpha kinases and PKR activity." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111905.
Full textAbstract:
Phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) on Serine 51 (Ser51) is an early event associated with downregulation of protein synthesis at the level of translation and constitutes a potent mechanism to overcome various stress conditions. In mammals, four eIF2alpha-kinases PERK, PKR, HRI and GCN2, activated following specific stresses, have been involved in this process. Our laboratory has previously demonstrated that the adaptor protein Nck, composed only of Src homology domains and classically implicated in cell signaling by activated plasma membrane receptor tyrosine kinases, modulates translation through its interaction with the beta-subunit of the eukaryotic initiation factor 2 (eIF2beta). Moreover, we reported that Nck-1 overexpression antagonizes the inhibition of translation in endoplasmic reticulum stress condition and prevents the PERK-mediated phosphorylation of the alpha-subunit of eIF2 on Ser51. In this thesis, I demonstrate that the adaptor protein Nck-1 modulates eIF2alpha-kinase-mediated eIF2alphaSer51 phosphorylation in a specific manner. More particularly, I show that Nck-1 overexpression reduces eIF2alpha phosphorylation in conditions activating PKR or HRI as described previously for PERK. In contrast, I observe that overexpression of Nck-1 in mammalian cells fails to attenuate eIF2alphaSer51 phosphorylation in response to amino acid starvation, a stress condition activating GCN2. I further confirm this observation by showing that Nck-1 fails to alter eIF2alphaSer51 phosphorylation in Saccharomyces cerevisiae, for which the sole eIF2alpha-kinase is GCN2. In addition, I report that Nck-1 reduces PKR activation in response to dsRNA. I also find that Nck-1 reduces dsRNA-induced activation of p38 MAPK, a PKR-downstream substrate, and cell death. Finally, I show that Nck-1 interacts exclusively with the inactivated form of PKR in a Src homology domain independent manner. All together these data uncover the existence of a novel mechanism regulating phosphorylation of eIF2alphaSer51 under various stress conditions and identifies Nck-1 as a modulator of the tumor suppressor and antiviral protein kinase PKR.
7
Rivière, Gwladys. "Étude par RMN de la créatine kinase musculaire et d’un nouveau domaine de liaison à l’ubiquitine dans la protéine STAM2." Thesis, Lyon 1, 2011. http://www.theses.fr/2011LYO10285/document.
Full textAbstract:
Au cours de cette thèse, nous avons étudié deux protéines par RMN : la créatine kinase musculaire (CK-MM) et le domaine UIM-SH3 de la protéine STAM2, seuls ou en interaction avec leurs partenaires. La CK-MM est une enzyme active sous forme dimérique. Elle appartient à la famille des guanidino-kinases et intervient dans le processus énergétique de la cellule. Le but de l’étude était d’élucider le mode de fonctionnement de la CK-MM. Pour cela, nous avons enregistré des expériences de relaxation R1, R2 et des expériences de perturbation de déplacement chimique sur la CK-MM libre et complexée avec MgADP et sous forme TSAC. Ces expériences montrent que la boucle 320s, spécifique à la reconnaissance des substrats, possède une dynamique rapide en absence de substrats et une dynamique ralentie en présence de substrats. La fixation des substrats dans les sites actifs de la CK-MM induit des modifications conformationelles importantes. La protéine STAM2 est composée de deux UBDs : VHS, et UIM et d’un domaine SH3 connu pour interagir avec des déubiquitinases UBPY et AMSH. Cette protéine est impliquée dans la voie de dégradation lysosomale. L’objectif de cette étude est la caractérisation du complexe SH3/ubiquitine. Pour cela, nous avons enregistré des expériences de perturbation de déplacement chimique et de relaxation R1, R2 et nOes sur le complexe UIM-SH3/ubiquitine. Ces expériences mettent en évidence que les domaines UIM et SH3 sont capables d’interagir chacun avec une ubiquitine, avec une affinité de l’ordre de la centaine de micromolaire. L’interface entre les UBDs et l’ubiquitine implique majoritairement des résidus hydrophobes et conservésIn this thesis, we study two proteins by NMR: the muscular creatine kinase (CK-MM) and the SH3 domain of STAM2 protein, in the free and complexed forms. CK-MM is an active homodimeric enzyme which belongs to the guanidino-phosphagen-kinase family. This enzyme is involved in energetic process in the cell. The aim of this study is to elucidate the functional mode of the CK-MM. For this purpose, we measured R1 and R2 relaxation rates and chemical shit perturbation experiments on the substrate-free CK-MM, the CK-MM/MgADP complex, and the inhibitory ternary complex CK-MM/MgADP-creatine-nitrate. The experiments show that the loop 320s, specific recognition of the substrates, possesses a fast dynamic in absence of substrates (in the order of nano-picosecond) and a slower dynamic in presence of creatine-MgADP-nitrate ion. The binding of the substrate in the two active sites induces of significant conformational modification of the CK-MM. STAM2 protein consists in two ubiquitin binding domains (VHS and UIM) and a SH3 domain which interacts with deubiquinating enzymes AMSH and UBPY. This protein is involved in the lysosomal degradation pathway. The aim of this study is the characterization of the interaction between SH3 domain of STAM2 and ubiquitin. For this, we recorded the R1, R2, nOes relaxation experiments and chemical shift perturbation experiments on the UIM-SH3/ubiquitin complex. These experiments show that SH3 and UIM domains interact each with a single ubiquitin, with affinity of the order of hundred micromolars. The interface between these UBDs and ubiquitin, involves mainly hydrophobic and conserved amino-acids
8
Dong, Shen. "Caractérisation de deux nouveaux mécanismes de régulation de l'initiation du signal induit par le récepteur pour l'antigène du lymphocyte T." Paris 6, 2009. http://www.theses.fr/2009PA066163.
Full textAbstract:
Mes travaux montrent que la stimulation du récepteur pour l’antigène du lymphocyte T induit la phosphorylation sur tyrosine et le recrutement à la protéine d’échafaudage LAT de l’adaptateur inhibiteur Dok-2. Ce dernier et son homologue Dok-1 sont alors caractérisés comme modulateurs du signal précoce induit par l’engagement du TCR et comme régulateurs du seuil d’activation du lymphocyte T. Par ailleurs, j’ai montré que les lymphocytes T CD4+ naïfs présentent une activation basale de Lck et Fyn qui n’est pas augmentée après stimulation du TCR. L’inhibition stable de l’expression de LAT par interférence d’ARN messagers diminue l’activation de Lck et Fyn et la phosphorylation des substrats directs de Lck. Ces défauts de signalisation sont corrigés lorsque le TCR est engagé fortement. Ainsi, mes données indiquent que l’engagement du TCR entraîne la stabilisation du pool de Lck actif auprès de ses substrats de façon dépendante de LAT et de la force de stimulation.
9
"Characterization of the interaction and phosphorylation mechanisms of serine/arginine-rich splicing factor 3 by SR protein kinase 2." 2013. http://library.cuhk.edu.hk/record=b5884372.
Full textAbstract:
Sou, Weng Hong.Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 96-106).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts also in Chinese.
10
Brammen, Christina Andrea Anna. "Kardiale Phänotypisierung einer transgenen Mauslinie mit herzspezifischer Calcium-Calmodulin-Kinase IIδc- Überexpression auf einem Phosphatase-Inhibitor-1- Knockout-Hintergrund." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0023-9632-2.
Full textBook chapters on the topic "SR Protein Specific Kinase 2"
1
"Non-specific protein-tyrosine kinase." In Class 2 Transferases, 441–565. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-540-85697-9_69.
Full text
2
"Non-specific serine/threonine protein kinase." In Class 2 Transferases, 1–123. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-540-85699-3_1.
Full text
3
Schechtman, Deborah, and Daria Mochly-Rosen. "Isozyme-Specific Inhibitors and Activators of Protein Kinase C." In Methods in Enzymology, 470–89. Elsevier, 2002. http://dx.doi.org/10.1016/s0076-6879(02)45039-2.
Full text
4
Burns-Hamuro, L. L., D. M. Barraclough, and S. S. Taylor. "Identification and Functional Analysis of Dual-Specific A Kinase-Anchoring Protein-2." In Methods in Enzymology, 354–74. Elsevier, 2004. http://dx.doi.org/10.1016/s0076-6879(04)90022-5.
Full text
5
JONES, E. G. "Cell-Specific Expression of a Multifunctional Protein Kinase in Primate Thalamic Relay Neurons." In Thalamic Networks for Relay and Modulation, 229–50. Elsevier, 1993. http://dx.doi.org/10.1016/b978-0-08-042274-9.50026-2.
Full text
6
Lymperopoulos, Anastasios, Giuseppe Rengo, Erhe Gao, Steven N. Ebert, Gerald W. Dorn, and Walter J. Koch. "Adrenal-Specific G Protein-Coupled Receptor Kinase (GRK)-2 Deficiency Reduces Circulating Catecholamine Levels and Improves Cardiac Function after Myocardial Infarction." In Catecholamine Research in the 21st Century, 207. Elsevier, 2014. http://dx.doi.org/10.1016/b978-0-12-800044-1.00186-0.
Full text
7
Winblad, Stefan, and Anne-Berit Ekström. "Myotonic Dystrophy." In Cognitive and Behavioral Abnormalities of Pediatric Diseases. Oxford University Press, 2010. http://dx.doi.org/10.1093/oso/9780195342680.003.0057.
Full textAbstract:
The myotonic dystrophies, type 1 (DM1) and 2 (DM2) are progressive, autosomal, dominantly inherited disorders, mainly characterized by muscle weakness and atrophy but also by a variable impact on heart, eye, brain, and the endocrine and the gastrointestinal system (Meola 2000). The worldwide prevalence is approximately 1 in 8,000. They are considered to be most common in Western Europe and Japan, but less prevalent in Southeast Asia, and rare or absent in southern and central Africa (Emery 1991). A prevalence of 18 in 340,000 children has been reported (Darin and Tulinius 2000). The cause of myotonic dystrophies is an unstable inherited repeat DNA expansions. Expansions are elements occurring and repeated throughout the human genome, typically polymorphic in the general population. Repeats can become unstable during DNA replication and, depending on specific repeat motif and location, expanded repeats can become pathogenic. In disease states, the number of repeats exceeds the normal range, leading to various pathogenic mechanisms (Ranum and Cooper 2006). DM1 is associated with an expanded (CTG)n repeat (>50 to several thousands) within the noncoding 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19q13.3. In DM2, another mutation exists, namely an expanded CCTG tetranucleotide repeat (from 75 to 11,000 repeats) in the first intron of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21 (Day and Ranum 2005). This means that two unrelated genes are associated with similar phenotypes although there are differences, including the age of onset and severity of symptoms (Meola 2000). The first signs of a DM2 disease are typically shown in adulthood, and no study has as yet systematically described cognitive or behavioral abnormalities in a childhood DM2 phenotype. Consequently, the following chapter focuses on a description of DM1. In this disorder, the age of onset is variable, meaning that there are congenital cases, as well as children, adults and patients experiencing the first symptoms very late in life. DM1 is traditionally divided into categories, each presenting with specific clinical features and broadly associated with the age of onset and extent of genetic abnormality.
Conference papers on the topic "SR Protein Specific Kinase 2"
1
Liu, Yuying, Adnan Al-Ayoubi, Hui Zheng, Jennifer Bethard, and Scott T. Eblen. "Abstract 3077: The SR protein kinase Clk1 phosphorylates SPF45 and regulates its localization, degradation and alternative splicing activity." In Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.am2011-3077.
Full text
2
Venkitachalam, Srividya, Fu-Yu Chueh, and Chao-Lan Yu. "Abstract 4008: Nuclear localization of Lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (LMO2) gene." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-4008.
Full text
3
Suga, K., Y. Uemura, T. Tsuijinaka, M. Sakon, J. Kambayashi, and T. Mori. "PROPERTIES OF PHOSPHATIDYLINOSITOL KINASE IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643807.
Full textAbstract:
We have reported the specific 32P-labelling in phosphatidyl-inositol-4-monophosphate(PIP) of intact platelets upon addition of the agents which elevate intracellular cAMP (Thrombos.Res.44, 155,1986).This event may be catalyzed by the action of Pl-kinase, the properties of which has not been elucidatedyet.Thereby, attempts were made to assay and to characterize PI-kinase of human platelets.Fresh lysed platelets prelabelled with 32P in cold Tris-HCl buffer containing 2mM EGTA were incubated at 37 C in the presence of MgCl2 for designated times and the phospholipids were extracted and analyzed by thin layer chromatography.32P-labelling in PIP was gradually increased in consort with the decreased labelling in PI-4,5-bisphosphate.As the changes in the labelling was not affected by the presence of apyrase and as the radioactive inositol trisphosphate was not detected,it was suggested that the changes is due to the action of phoshomono-esterase rather than PI-kinase or phospholipase C.When 32P-ATP was added to non-labelled lysed platelets upon incubation, 32P was labelled only into PIP and the amount was markedly increased until 5min. after incubation.Since the labelling was strongly inhibited by apyrase,it likely reflects the activity of Pl-kinase. The activity of PI-kinase thus measured required Mg2+ strictly for the activity and the maximal activity was obtained in the presence of 30mM Mg2+ .In contrast,it was markedly inhibited in the presence of Ca2+ (as low as 2mM Ca2+ in the presence of 2mM EGTA),which was compatible.with our previous findings with intact platelets. The activity of A-kinase was not inhibited by a low concentration of Ca2+ .Furthermore,the activity was inhibited by cAMP or dbcAMP in a dose related manner and no enhancement of the activity Was obtained by the addition of catalytic subunit of A-kinase,though a significant reduction in the activity was observed in the presence of inhibitor protein to A-kinase. From these observations,the following conclusions were obtained; l)The activity of Pl-kinase in lysed platelets may be determined by pulse labelling with 32P-ATP. 2)It requires Mg2+ absolutely and is inhibited by a very low concentration of Ca2+. 3)Pl-kinase is activated by A-kinase but the activated enzyme is inhibited by cAMP, suggesting the presence of feedback mechanism.
4
SIMON, M. F., H. CHAP, and L. DOUSTE-BLAZY. "EFFECTS OF SIN 1 ON PLATELET ACTIVATION INDUCED BY THROMBIN IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643423.
Full textAbstract:
The mechanism of platelet activation is well known. The interaction of agonist such as thrombin, on specific membrane receptor induces phosphatidylinositol-specific phospholipase C activation, with a concomitant formation of two second messengers (from PIP2): inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is able to induce a rapid discharge of Ca2+ from internal stores and Ca2+ influx through plasma membrane by unidentified Ca2+ channels linked to receptor activation. The increase of cytoplasmic free calcium concentration leads to the activation of the calcium calmodulin dependent myosine light chain kinase which phosphoryla-tes 20 kD proteins (myosine light chain). DAG is a potent activator of protein kinase C, which phosphorylates 40 kD proteins. These different pathways act in synergism.Sin 1 is a platelet aggregating inhibitor. This compound is an active metabolite of molsidomine, which activates platelet guany-late cyclase, inducing a rapid rise in cyclic GMP level. The precise role of cyclic GMP in platelet activation is not yet known. In order to study the mechanism of action of this drug, we tried to determine the effect of Sin 1 on the different steps described above. We measured Ca2+ fluxes and phospholipase C activation in thrombin (0,5 U/ml) stimulated platelets in the presence of different doses of Sin 1 (10™7-10™3M). Serotonin secretion was inhibited by 30 % with Sin 1 (10™4M-10™5m). A parallel inhibition of phospholipase C was detected by measurement of [32P)-PA level. Platelets loaded with Quin 2 and stimulated by thrombin showed a 70 % inhibition of external Ca2+ influx as soon as a concentration of 10™7M of Sin 1 was added. A study on platelet loaded with [45Ca2+) and Quin 2 confirmed these results. On the contrary, discharge of internal Ca2+ store seemed to be unaffected.In conclusion, the major effect of Sin 1 on platelet phospholipase C pathway is an inhibition of Ca2+ influx through plasma membrane. Some further experiments are necessary to shown whether this inhibition is correlated with cyclic GMP formation (the major effect of Sin 1) and try to establish a relation between this inhibition and that exerted on phospholipase C.Sin 1 was a generous gift of Hoechst.
5
Sakon, M., Y. Uemura, K. Suga, T. Tsujinaka, J. Kambayashi, and T. Mori. "STUDIES ON PHOSPHATASES IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644494.
Full textAbstract:
Activation of platelets by various agonists has been ascribed to be associated with phosphorylation and dephosphorylation of specific proteins such as 20K and 47K polypeptide. Although protein kinases such as myosin light chain kinase and C kinase have been extensively studied, little information is currently available on platelet phosphatases, which may play a crucial role in the regulation of stimulus-linked protein phosphorylation. Thereby, the present study was conducted to know some characters of platelet phosphatases. Glycerol loaded platelets prepared from human platelet concentrates were subjected to osmotic lysis in 20 mM HEPES-NaOH buffer containing 5 mM EDTA, 0.5 mM dithio-threitol and various protease inhibitors and a soluble fraction was obtained by centrifugation, The activity of phosphatase was assayed at pH 7.35, using paranitrophenylphosphate as a substrate. Leupeptin and EDTA were added to the reaction mixture to avoid proteolytic attack to the enzyme. The neutral phosphatase was partially purified from the soluble fraction by a combination of ammonium sulfate fractionation and column chromatographies. Five distinct peaks with neutral phosphatase activity were obtained by a linear gradient elution ( 0−0.5 M KCl ) in DEAE Sepharose CL-6B of 0−60 % ammonium sulfate precipitate. The phosphatase activity of one peak eluted at 0.2M KCl was maximum at pH below 6, which was considered to be acid phosphatase, and the remaining four peaks' optimal pH was between 7.0−7.5. These four peaks were termed as PH-I (passed through fraction), PH-II (0.1M KCl), PH-III (0.25M KCl) and PH-IV (0.3M KCl). The respective peak was eluted as a single peak on Ultrigel AcA 34 and the molecular weight was estmated as follows; I-55K, II-40K, III-55K, IV-37K. PH-I − II were active in the presnce of EDTA and were not affect ed by divalent cations (Mg++ , Mn++ , Ca++ ) , whereas PH-III was highly dependent upon Mg++. The activity of PH-IV was completely dependent on Mn++. From these observations, the following conclusions were obtained; (1) Human platelets contain four species of neutral phosphatases, in addition to acid phosphatase. (2) Each neutral phosphatase is distincive by molecular weight and requirement of divalent cations.
6
Schleuning, W. D. "THE BIOCHEMISTRY AND CELL BIOLOGY OF SINGLE CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642956.
Full textAbstract:
Urokinase was discovered in the late nineteenth century, as an enzymatic principle in urine, that initiates the dissolution of blood clots. The basis of this phenomenon was recognized more than fifty years ago as the activation of plasminogen, the precursor of a tryptic protease, then known as profibrinolysin. Despite this long history, detailed data on the biochemistry of plasminogen activation have only become available recently. Urokinase (now designated urokinase-type plasminogen activator : u-PA) is synthesized and secreted as a single chain polypeptide (Mr-: 53,000) by many cell types. Single chain u-PA (scu-PA) is with equal justification called prourokinase (pro-u-PA), notwithstanding its low catalytic activity for synthetic peptide substrates and plasminogen, as most proenzymes of proteases display a certain degree of activity. The structure of pro-u-PA has been elucidated by protein and cDNA sequencing. It consists of three domains, exhibiting characteristic homology to other proteins: a serine protease domain, homologous to trypsin, chymotrypsin and elastase; a kringle domain, likewise found in prothrombin, plasminogen, tissue-type plasminogen activator (t-PA) and Factor XII; and an epidermal growth factor (EGF)-like domain, found in many other proteins, including certain clotting factors. Pro-u-PA is activated by the cleavage of its LYS158-Ile159 h1 bY either plasmin or kallikrein. This cleavage leads to a high increase of Kcat values with respect to both plasminogen and synthetic peptide substrates, but apparently to a reduction of its affinity to plasminogen. Thrartoin inactivates pro-u-PA irreversibly by the cleavage of the Arg156-Phe157 bond. U-PA but not pro-u-PA rapidly forms ccnplexes with plasminogen activator inhibitors (PAI)-l and PAI-2: second order rate constants Kass are respectively > 107 and 0.9xl06 (M-11sec-1). Unknown enzymes process pro-u-PA and u-PA to low molecular weight (LMW) pro-u-PA and LMW u-PA (Mr: 33,000) by cutting off a fragment consisting of the kr ingle and the EGF—like region. Pro—u—PA mediated plasminogen activation is fibrin dependent in vivo, and to a certain degree in vitro. Hie biochemical basis of this fibrin specificity is at present uncertain, although there are reports indicating that it may require polyvalent cations. Through its EGF-like region HMW pro-u-PA and HMW u-PA are capable of binding to specific membrane protein receptors which are found on many cells. Thus, u-PA activity may be restricted to the cell surface. According to a recent report, binding of u—PA to the receptor may also mediate signal transduction in auto- or paracrine growth control. In cells permissive for the respective pathways, pro-u-PA gene transcription is stimulated by mechanisms of signal transduction, that include the cAMP, the tyrosine specific kinase and the protein kinase C dependent pathways. Glucocorticoid hormones downregulate pro-u-PA gene transcription in cells where the gene is canstitutively expressed. Although different cells vary greatly in their response to agents that stimulate urokinase biosynthesis, growth factors and other mitogens are in many cases effective inducers. Significantly elevated levels of u-PA are also found in many malignant tissues. These findings and many others suggest that plasminogen activation by u-PA provides localized extracellular matrix degradation which is required for invasive growth, cell migration and other forms of tissue remodelling. Fibrin represents in this view only a variant of an extracellular matrix, which is provided through the clotting system in the case of an emergency.
7
Lapetina, Eduardo G. "THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644775.
Full textAbstract:
It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.
Reports on the topic "SR Protein Specific Kinase 2"
1
Barash, Itamar, and Robert Rhoads. Translational Mechanisms Governing Milk Protein Levels and Composition. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7696526.bard.
Full textAbstract:
Original objectives: The long-term goal of the research is to achieve higher protein content in the milk of ruminants by modulating the translational apparatus of the mammary gland genetically, nutritionally, or pharmacologically. The short-term objectives are to obtain a better understanding of 1) the role of amino acids (AA) as regulators of translation in bovine and mouse mammary epithelial cells and 2) the mechanism responsible for the synergistic enhancement of milk-protein mRNA polyadenylation by insulin and prolactin. Background of the topic: In many cell types and tissues, individual AA affect a signaling pathway which parallels the insulin pathway to modulate rates and levels of protein synthesis. Diverse nutritional and hormonal conditions are funneled to mTOR, a multidomain serine/threonine kinase that regulates a number of components in the initiation and elongation stages of translation. The mechanism by which AA signal mTOR is largely unknown. During the current grant period, we have studied the effect of essential AA on mechanisms involved in protein synthesis in differentiated mammary epithelial cells cultured under lactogenic conditions. We also studied lactogenic hormone regulation of milk protein synthesis in differentiated mammary epithelial cells. In the first BARD grant (2000-03), we discovered a novel mechanism for mRNA-specific hormone-regulated translation, namely, that the combination of insulin plus prolactin causes cytoplasmic polyadenylation of milk protein mRNAs, which leads to their efficient translation. In the current BARD grant, we have pursued the signaling pathways of this novel hormone action. Major conclusions/solutions/achievements: The positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediates the synthesis rates of total and specific milk proteins in mammary epithelial cells. The current in vitro study revealed cryptic negative effects of Lys, His, and Thr on cellular mechanisms regulating translation initiation and protein synthesis in mammary epithelial cells that could not be detected by conventional in vivo analyses. We also showed that a signaling pathway involving Jak2 and Stat5, previously shown to lead from the prolactin receptor to transcription of milk protein genes, is also used for cytoplasmic polyadenylation of milk protein mRNAs, thereby stabilizing these mRNAs and activating them for translation. Implications: In vivo, plasma AA levels are affected by nutritional and hormonal effects as well as by conditions of exercise and stress. The amplitude in plasma AA levels resembles that applied in the current in vitro study. Thus, by changing plasma AA levels in the epithelial cell microenvironment or by sensitizing the mTOR pathway to their presence, it should be possible to modulate the rate of milk protein synthesis. Furthermore, knowledge that phosphorylation of Stat5 is required for enhanced milk protein synthesis in response to lactogenic opens the possibility for pharmacologic approaches to increase the phosphorylation of Stat5 and, thereby, milk protein production.
2
Avni, Adi, and Gitta L. Coaker. Proteomic investigation of a tomato receptor like protein recognizing fungal pathogens. United States Department of Agriculture, January 2015. http://dx.doi.org/10.32747/2015.7600030.bard.
Full textAbstract:
Maximizing food production with minimal negative effects on the environment remains a long-term challenge for sustainable food production. Microbial pathogens cause devastating diseases, minimizing crop losses by controlling plant diseases can contribute significantly to this goal. All plants possess an innate immune system that is activated after recognition of microbial-derived molecules. The fungal protein Eix induces defense responses in tomato and tobacco. Plants recognize Eix through a leucine-rich-repeat receptor- like-protein (LRR-RLP) termed LeEix. Despite the knowledge obtained from studies on tomato, relatively little is known about signaling initiated by RLP-type immune receptors. The focus of this grant proposal is to generate a foundational understanding of how the tomato xylanase receptor LeEix2 signals to confer defense responses. LeEix2 recognition results in pattern triggered immunity (PTI). The grant has two main aims: (1) Isolate the LeEix2 protein complex in an active and resting state; (2) Examine the biological function of the identified proteins in relation to LeEix2 signaling upon perception of the xylanase elicitor Eix. We used two separate approaches to isolate receptor interacting proteins. Transgenic tomato plants expressing LeEix2 fused to the GFP tag were used to identify complex components at a resting and activated state. LeEix2 complexes were purified by mass spectrometry and associated proteins identified by mass spectrometry. We identified novel proteins that interact with LeEix receptor by proteomics analysis. We identified two dynamin related proteins (DRPs), a coiled coil – nucleotide binding site leucine rich repeat (SlNRC4a) protein. In the second approach we used the split ubiquitin yeast two hybrid (Y2H) screen system to identified receptor-like protein kinase At5g24010-like (SlRLK-like) (Solyc01g094920.2.1) as an interactor of LeEIX2. We examined the role of SlNRC4a in plant immunity. Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. We propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 inVHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune . The interaction of LeEIX2 with SlRLK-like was verified using co- immunoprecipitation and a bimolecular fluorescence complementation assay. The defence responses induced by EIX were markedly reduced when SlRLK-like was over-expressed, and mutation of slrlk-likeusing CRISPR/Cas9 increased EIX- induced ethylene production and SlACSgene expression in tomato. Co-expression of SlRLK-like with different RLPs and RLKs led to their degradation, apparently through an endoplasmic reticulum-associated degradation process. We provided new knowledge and expertise relevant to expression of specific be exploited to enhance immunity in crops enabling the development of novel environmentally friendly disease control strategies.
3
Sessa, Guido, and Gregory Martin. MAP kinase cascades activated by SlMAPKKKε and their involvement in tomato resistance to bacterial pathogens. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7699834.bard.
Full textAbstract:
The research problem: Pseudomonas syringae pv. tomato (Pst) and Xanthomonas campestrispv. vesicatoria (Xcv) are the causal agents of tomato bacterial speck and spot diseases, respectively. These pathogens colonize the aerial parts of the plant and cause economically important losses to tomato yield worldwide. Control of speck and spot diseases by cultural practices or chemicals is not effective and genetic sources of resistance are very limited. In previous research supported by BARD, by gene expression profiling we identified signaling components involved in resistance to Xcvstrains. Follow up experiments revealed that a tomato gene encoding a MAP kinase kinase kinase (MAPKKKe) is required for resistance to Xcvand Pststrains. Goals: Central goal of this research was to investigate the molecular mechanisms by which MAPKKKεand associated MAP kinase cascades regulate host resistance. Specific objectives were to: 1. Determine whether MAPKKKεplays a broad role in defense signaling in plants; 2. Identify components of MAP kinase cascades acting downstream of MAPKKKε; 3. Determine the role of phosphorylation-related events in the function of MAPKKKε; 4. Isolate proteins directly activated by MAPKKKε-associatedMAPK modules. Our main achievements during this research program are in the following major areas: 1. Characterization of MAPKKKεas a positive regulator of cell death and dissection of downstream MAP kinase cascades (Melech-Bonfil et al., 2010; Melech-Bonfil and Sessa, 2011). The MAPKKKεgene was found to be required for tomato resistance to Xcvand Pstbacterial strains and for hypersensitive response cell death triggered by different R gene/effector gene pairs. In addition, overexpression analysis demonstrated that MAPKKKεis a positive regulator of cell death, whose activity depends on an intact kinase catalytic domain. Epistatic experiments delineated a signaling cascade downstream of MAPKKKεand identified SIPKK as a negative regulator of MAPKKKε-mediated cell death. Finally, genes encoding MAP kinase components downstream of MAPKKKεwere shown to contribute to tomato resistance to Xcv. 2. Identification of tomato proteins that interact with MAPKKKεand play a role in plant immunity (Oh et al., 2011). We identified proteins that interact with MAPKKKε. Among them, the 14-3-3 protein TFT7 was required for cell death mediated by several R proteins. In addition, TFT7 interacted with the MAPKK SlMKK2 and formed homodimersin vivo. Thus, TFT7 is proposed to recruit SlMKK2 and MAPKKK client proteins for efficient signal transfer. 3. Development of a chemical genetic approach to identify substrates of MAPKKKε-activated MAP kinase cascades (Salomon et al., 2009, 2011). This approach is based on engineering the kinase of interest to accept unnatural ATP analogs. For its implementation to identify substrates of MAPKKKε-activated MAP kinase modules, we sensitized the tomato MAP kinase SlMPK3 to ATP analogs and verified its ability to use them as phosphodonors. By using the sensitized SlMPK3 and radiolabeled N6(benzyl)ATP it should be possible to tag direct substrates of this kinase. 4. Development of methods to study immunity triggered by pathogen-associated molecular patterns (PAMPs) in tomato and N. benthamiana plants (Kim et al., 2009; Nguyen et al. 2010). We developed protocols for measuring various PTI-associatedphenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition at the cell wall, activation of MAP kinases, and a luciferase-based reporter system for use in protoplasts. Scientific and agricultural significance: Our research activities discovered and characterized a signal transduction pathway mediating plant immunity to bacterial pathogens. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease. In addition, we successfully developed new biochemical and molecular methods that can be implemented in the study of plant immunity and other aspects of plant biology.
4
Lers, Amnon, and Gan Susheng. Study of the regulatory mechanism involved in dark-induced Postharvest leaf senescence. United States Department of Agriculture, January 2009. http://dx.doi.org/10.32747/2009.7591734.bard.
Full textAbstract:
Postharvest leaf senescence contributes to quality losses in flowers and leafy vegetables. The general goal of this research project was to investigate the regulatory mechanisms involved in dark-induced leaf senescence. The regulatory system involved in senescence induction and control is highly complex and possibly involves a network of senescence promoting pathways responsible for activation of the senescence-associated genes. Pathways involving different internal signals and environmental factors may have distinctive importance in different leaf senescence systems. Darkness is known to have a role in enhancement of postharvest leaf senescence and for getting an insight into its regulatory mechanism/s we have applied molecular genetics and functional genomics approaches. The original objectives were: 1. Identification of dark-induced SAGs in Arabidopsis using enhancer/promoter trap lines and microarray approaches; 2. Molecular and functional characterization of the identified genes by analyzing their expression and examining the phenotypes in related knockout mutant plants; 3. Initial studies of promoter sequences for selected early dark-induced SAGs. Since genomic studies of senescence, with emphasis on dark-induced senescence, were early-on published which included information on potential regulatory genes we decided to use this new information. This is instead of using the uncharacterized enhancer/promoter trap lines as originally planned. We have also focused on specific relevant genes identified in the two laboratories. Based on the available genomic analyses of leaf senescence 10 candidate genes hypothesized to have a regulatory role in dark-induced senescence were subjected to both expression as well as functional analyses. For most of these genes senescence-specific regulation was confirmed, however, functional analyses using knock-out mutants indicated no consequence to senescence progression. The transcription factor WARK75 was found to be specifically expressed during natural and dark-induced leaf senescence. Functional analysis demonstrated that in detached leaves senescence under darkness was significantly delayed while no phenotypic consequences could be observed on growth and development, including no effect on natural leaf senescence,. Thus, WARKY75 is suggested to have a role in dark-induced senescence, but not in natural senescence. Another regulatory gene identified to have a role in senescence is MKK9 encoding for a Mitogen-Activated Protein Kinase Kinase 9 which is upregulated during senescence in harvested leaves as well as in naturally senescing leaves. MKK9 can specifically phosphorylate another kinase, MPK6. Both knockouts of MKK9 and MPK6 displayed a significantly senescence delay in harvested leaves and possibly function as a phosphorelay that regulates senescence. To our knowledge, this is the first report that clearly demonstrates the involvement of a MAP kinase pathway in senescence. This research not only revealed a new signal transduction pathway, but more important provided significant insights into the regulatory mechanisms underlying senescence in harvested leaves. In an additional line of research we have employed the promoter of the senescence-induced BFN1 gene as a handle for identifying components of the regulatory mechanism. This gene was shown to be activated during darkinduced senescence of detached leaves, as well as natural senescence. This was shown by following protein accumulation and promoter activity which demonstrated that this promoter is activated during dark-induced senescence. Analysis of the promoter established that, at least some of the regulatory sequences reside in an 80 bps long fragment of the promoter. Overall, progress was made in identification of components with a role in dark-induced senescence in this project. Further studies should be done in order to better understand the function of these components and develop approaches for modulating the progress of senescence in crop plants for the benefit of agriculture.
5
Sessa, Guido, and Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695876.bard.
Full textAbstract:
The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
6
Delmer, Deborah P., Douglas Johnson, and Alex Levine. The Role of Small Signal Transducing Gtpases in the Regulation of Cell Wall Deposition Patterns in Plants. United States Department of Agriculture, August 1995. http://dx.doi.org/10.32747/1995.7570571.bard.
Full textAbstract:
The combined research of the groups of Delmer, Levine and Johnson has led to a number of interesting findings with respect to the function of the small GTPase Rac in plants and also opened up new leads for future research. The results have shown: 1) The Rac13 protein undergoes geranylgeranlyation and is also translocated to the plasma membrane as found for Rac in mammals; 2) When cotton Rac13 is highly- expressed in yeast, it leads to an aberrant phenotype reminiscent of mutants impaired in actin function, supporting a role for Rac13 in cytoskeletal organization; 3) From our searches, there is no strong evidence that plants contain homologs of the related CDC42 genes found in yeast and mammals; 4) We have identified a rather unique Rac gene in Arabidopsis that has unusual extensions at both the N- and C-terminal portions of the protein; 5) New evidence was obtained that an oxidative burst characterized by substantial and sustained production of H202 occurs coincident with the onset of secondary wall synthesis in cotton fibers. Further work indicates that the H202 produced may be a signal for the onset of this phase of development and also strongly suggests that Rac plays an important role in signaling for event. Since the secondary walls of plants that contain high levels of lignin and cellulose are the major source of biomass on earth, understanding what signals control this process may well in the future have important implications for manipulating the timing and extent of secondary wall deposition. 6) When the cotton Rac13 promoter is fused to the reporter gene GUS, expression patterns in Arabidopsis indicate very strong and specific expression in developing trichomes and in developing xyelm. Since both of these cell types are engaged in secondary wall synthesis, this further supports a role for Rac in signaling for onset of this process. Since cotton fibers are anatomically defined as trichomes, these data may also be quite useful for future studies in which the trichomes of Arabidopsis may serve as a model for cotton fiber development; the Rac promoter can therefore be useful to drive expression of other genes proposed to affect fiber development and study the effects on the process; 7) The Rac promoter has also been shown to be the best so far tested for use in development of a system for transient transformation of developing cotton fibers, a technique that should have many applications in the field of cotton biotechnology; 8) One candidate protein that may interact with Rac13 to be characterized further in the future is a protein kinase that may be analogous to the PAK kinase that is known to interact with Rac in mammals.
7
Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.
Full textAbstract:
The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.
8
Prusky, Dov, Noel Keen, and John Browse. Modulation of the synthesis of the main preformed antifungal compound as abasis for the prevention of postharvest disease of C. gloeosporioides in avocado fruits. United States Department of Agriculture, December 2001. http://dx.doi.org/10.32747/2001.7575273.bard.
Full textAbstract:
The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned , The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned D9 and D12 desaturase, a protein kinase and a elongase that their transcriptional activation is significantly enhanced during the enhanced synthesis of the antifungal diene. Although we are far away from a complete elucidation of the synthesis of the antifungal compound we have stepped forward determining some of the key steps that might be involved in its synthesis.
9
Hansen, Peter J., and Amir Arav. Embryo transfer as a tool for improving fertility of heat-stressed dairy cattle. United States Department of Agriculture, September 2007. http://dx.doi.org/10.32747/2007.7587730.bard.
Full textAbstract:
The overall objective of the current proposal is to develop procedures to improve the pregnancy rate achieved following transfer of fresh or cryopreserved embryos produced in the laboratory into heat-stress recipients. The overall hypothesis is that pregnancy rate in heat-stressed lactating cows can be improved by use of embryo transfer and that additional gains in pregnancy rate can be achieved through development of procedures to cryopreserve embryos, select embryos most likely to establish and maintain pregnancy after transfer, and to enhance embryo competence for post-transfer survival through manipulation of culture conditions. The original specific objectives were to 1) optimize procedures for cryopreservation (Israel/US), 2) develop procedures for identifying embryos with the greatest potential for development and survival using the remote monitoring system called EmbryoGuard (Israel), 3) perform field trials to test the efficacy of cryopreservation and the EmbryoGuard selection system for improving pregnancy rates in heat-stressed, lactating cows (US/Israel), 4) test whether selection of fresh or frozen-thawed blastocysts based on measurement of group II caspase activity is an effective means of increasing survival after cryopreservation and post-transfer pregnancy rate (US), and 5) identify genes in blastocysts induced by insulin-like growth factor-1 (IGF-1) (US). In addition to these objectives, additional work was carried out to determine additional cellular determinants of embryonic resistance to heat shock. There were several major achievements. Results of one experiment indicated that survival of embryos to freezing could be improved by treating embryos with cytochalasin B to disrupt the cytoskeleton. An additional improvement in the efficacy of embryo transfer for achieving pregnancy in heat-stressed cows follows from the finding that IGF-1 can improve post-transfer survival of in vitro produced embryos in the summer but not winter. Expression of several genes in the blastocyst was regulated by IGF-1 including IGF binding protein-3, desmocollin II, Na/K ATPase, Bax, heat shock protein 70 and IGF-1 receptor. These genes are likely candidates 1) for developing assays for selection of embryos for transfer and 2) as marker genes for improving culture conditions for embryo production. The fact that IGF-1 improved survival of embryos in heat-stressed recipients only is consistent with the hypothesis that IGF-1 confers cellular thermotolerance to bovine embryos. Other experiments confirmed this action of IGF-1. One action of IGF-1, the ability to block heat-shock induced apoptosis, was shown to be mediated through activation of the phosphatidylinositol 3-kinase pathway. Other cellular determinants of resistance of embryos to elevated temperature were identified including redox status of the embryo and the ceramide signaling pathway. Developmental changes in embryonic apoptosis responses in response to heat shock were described and found to include alterations in the capacity of the embryo to undergo caspase-9 and caspase-3 activation as well as events downstream from caspase-3 activation. With the exception of IGF-1, other possible treatments to improve pregnancy rate to embryo transfer were not effective including selection of embryos for caspase activity, treatment of recipients with GnRH.and bilateral transfer of twin embryos. In conclusion, accomplishments achieved during the grant period have resulted in methods for improving post-transfer survival of in vitro produced embryos transferred into heat-stressed cows and have lead to additional avenues for research to increase embryo resistance to elevated temperature and improve survival to cryopreservation. In addition, embryo transfer of vitrified IVF embryos increased significantly the pregnancy rate in repeated breeder cows.
10
McClure, Michael A., Yitzhak Spiegel, David M. Bird, R. Salomon, and R. H. C. Curtis. Functional Analysis of Root-Knot Nematode Surface Coat Proteins to Develop Rational Targets for Plantibodies. United States Department of Agriculture, October 2001. http://dx.doi.org/10.32747/2001.7575284.bard.
Full textAbstract:
The goal of this research was to provide a better understanding of the interface between root-knot nematodes, Meloidogyne spp., and their host in order to develop rational targets for plantibodies and other novel methods of nematode control directed against the nematode surface coat (SC). Specific objectives were: 1. To produce additional monoclonal SC antibodies for use in Objectives 2, 3, and 4 and as candidates for development of plantibodies. 2. To determine the production and distribution of SC proteins during the infection process. 3. To use biochemical and immunological methods to perturbate the root-knot nematode SC in order to identify SC components that will serve as targets for rationally designed plantibodies. 4. To develop SC-mutant nematodes as additional tools for defining the role of the SC during infection. The external cuticular layer of nematodes is the epicuticle. In many nematodes, it is covered by a fuzzy material termed "surface coat" (SC). Since the SC is the outermost layer, it may playa role in the interaction between the nematode and its surroundings during all life stages in soil and during pathogenesis. The SC is composed mainly of proteins, carbohydrates (which can be part of glycoproteins), and lipids. SC proteins and glycoproteins have been labeled and extracted from preparasitic second-stage juveniles and adult females of Meloidogyne and specific antibodies have been raised against surface antigens. Antibodies can be used to gain more information about surface function and to isolate genes encoding for surface antigens. Characterization of surface antigens and their roles in different life-stages may be an important step towards the development of alternative control. Nevertheless, the role of the plant- parasitic nematode's surface in plant-nematode interaction is still not understood. Carbohydrates or carbohydrate-recognition domains (CROs) on the nematode surface may interact with CROs or carbohydrate molecules, on root surfaces or exudates, or be active after the nematode has penetrated into the root. Surface antigens undoubtedly play an important role in interactions with microorganisms that adhere to the nematodes. Polyclonal (PC) and monoclonal (MC) antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes, were used to characterize the surface coat and secreted-excreted products of M. javanica and M. incognita. Some of the MC and PC antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Further characterization, in planta, of the epitopes recognized by the antibodies, showed that they were present in the parasitic juvenile stages and that the surface coat is shed during root penetration by the nematode and its migration between root cells. At the molecular level, we have followed two lines of experimentation. The first has been to identify genes encoding surface coat (SC) molecules, and we have isolated and characterized a small family of mucin genes from M. incognita. Our second approach has been to study host genes that respond to the nematode, and in particular, to the SC. Our previous work has identified a large suite of genes expressed in Lycopersicon esculentum giant cells, including the partial cDNA clone DB#131, which encodes a serine/threonine protein kinase. Isolation and predicted translation of the mature cDNA revealed a frame shift mutation in the translated region of nematode sensitive plants. By using primers homologous to conserved region of DB#131 we have identified the orthologues from three (nematode-resistant) Lycopersicon peruvianum strains and found that these plants lacked the mutation.